Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping
2016; Wiley; Volume: 78; Issue: 2 Linguagem: Inglês
10.1002/mrm.26379
ISSN1522-2594
AutoresTroels Juul, Fredrik Palm, Per Mose Nielsen, Lotte Bonde Bertelsen, Christoffer Laustsen,
Tópico(s)NMR spectroscopy and applications
ResumoMagnetic Resonance in MedicineVolume 78, Issue 2 p. 457-461 NoteFree Access Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping Troels Juul, Troels Juul MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorFredrik Palm, Fredrik Palm Department of Medical Cell Biology, Uppsala University, Uppsala, SwedenSearch for more papers by this authorPer Mose Nielsen, Per Mose Nielsen MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorLotte Bonde Bertelsen, Lotte Bonde Bertelsen MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorChristoffer Laustsen, Corresponding Author Christoffer Laustsen [email protected] MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkCorrespondence to: Christoffer Laustsen, Ph.D., Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark. E-mail: [email protected]; Twitter: @chris_laustsenSearch for more papers by this author Troels Juul, Troels Juul MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorFredrik Palm, Fredrik Palm Department of Medical Cell Biology, Uppsala University, Uppsala, SwedenSearch for more papers by this authorPer Mose Nielsen, Per Mose Nielsen MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorLotte Bonde Bertelsen, Lotte Bonde Bertelsen MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorChristoffer Laustsen, Corresponding Author Christoffer Laustsen [email protected] MR Research Centre, Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkCorrespondence to: Christoffer Laustsen, Ph.D., Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark. E-mail: [email protected]; Twitter: @chris_laustsenSearch for more papers by this author First published: 16 August 2016 https://doi.org/10.1002/mrm.26379Citations: 3AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Purpose It has been demonstrated that hyperpolarized 13C MR is a useful tool to study cultured cells. However, cells in culture can alter phenotype, which raises concerns regarding the in vivo significance of such findings. Here we investigate if metabolic phenotyping using hyperpolarized 13C MR is suitable for cells isolated from kidney tissue, without prior cell culture. Methods Isolation of tubular cells from freshly excised kidney tissue and treatment with either ouabain or antimycin A was investigated with hyperpolarized MR spectroscopy on a 9.4 Tesla preclinical imaging system. Results Isolation of tubular cells from less than 2 g of kidney tissue generally resulted in more than 10 million live tubular cells. This amount of cells was enough to yield robust signals from the conversion of 13C-pyruvate to lactate, bicarbonate and alanine, demonstrating that metabolic flux by means of both anaerobic and aerobic pathways can be quantified using this technique. Conclusion Ex vivo metabolic phenotyping using hyperpolarized 13C MR in a preclinical system is a useful technique to study energy metabolism in freshly isolated renal tubular cells. This technique has the potential to advance our understanding of both normal cell physiology as well as pathological processes contributing to kidney disease. Magn Reson Med 78:457–461, 2017. © 2016 International Society for Magnetic Resonance in Medicine INTRODUCTION Dissolution dynamic nuclear polarization is a method for hyperpolarizing nuclear spins for MR spectroscopy (MRS) to a degree that yields a >10,000-fold increase in signal-to-noise ratio compared with conventional MRS 1. This enables the use of hyperpolarized 13C in biologically relevant tracer molecules and to trace the conversion to downstream metabolites with distinct chemical shifts. However, the tracer molecules are limited by the intrinsic T1 relaxation decay of the hyperpolarized spin system 2, 3. Thus, the metabolic conversion of interest must occur within a few minutes. [1-13C]Pyruvate has been widely used as a tracer due to its chemical and physical properties that are especially suited for hyperpolarization and its rapid cellular uptake and subsequent conversion into metabolites as it enters different metabolic pathways 4, 5. Pyruvate is metabolized to lactate by the enzyme lactate dehydrogenase (LDH) in the anaerobic fermentation step following glycolysis, whereas it is metabolized to acetyl-CoA by pyruvate dehydrogenase complex (PDH) in the mitochondrial matrix as it enters the Krebs' cycle 6, 7. The balance between LDH and PDH activity, therefore, reflects the metabolic balance between anaerobic and aerobic energy metabolism, respectively. When using [1-13C]pyruvate, the PDH-catalyzed reaction is measured indirectly, as the hyperpolarized C-1-carbon is incorporated into the concomitantly produced carbon dioxide, which is in equilibrium with MR-detectable bicarbonate 8. Furthermore, pyruvate can also be metabolized to alanine by the enzyme alanine aminotransferase (ALT), linking carbohydrate and amino acid metabolism 6, 7. Several studies have used hyperpolarized MRS both in vitro 9-13 and in vivo 5, 14-16, including the first clinical trial investigating patients with prostate cancer 17. In that study, Keshari et al used hyperpolarized MRS to study prostate tissue slices ex vivo 18, but, to the best of our knowledge, no study has yet been performed directly on type-specific cells isolated from kidney tissue. In this study, we report a method for detection and quantification of metabolic pathways using hyperpolarized MRS in rodent renal tubular cells freshly isolated without prior in vitro cultivation. The results demonstrate that hyperpolarized MRS is a promising novel technique to study cellular metabolism. METHODS Animals The study included eight Wistar rats (Taconic, Ry, Denmark). All animals had free access to water and standard chow throughout the study. The rats were kept in cages with a 12:12-h light–dark cycle, a temperature of 21 ± 2°C and a humidity of 55 ± 5%. The study complied with the guidelines for the care and use of laboratory animals and was approved by the Danish Inspectorate of Animal Experiments. Isolation of Renal Tubular Cells For each isolation, the renal cortical tissue of one Wistar rat was used. The rat was anesthetized (3% Servoflurane in air) and both kidneys were excised and placed in a plastic tray on ice containing respiration media [in mM: 113 NaCl, 4 KCl, 27.2 NaHCO3, 1 KH2PO4, 1.2 MgCl2, 1 CaCl2, 10 HEPES (Sigma-Aldrich, Brøndby, DK), 5.8 glucose, 0.5 Ca-lactate, 2 glutamine (Acros Organics, Geel, Belgium) and 0.5 mg/L streptomycin (Sigma-Aldrich)], with pH adjusted to 7.4 with NaOH (Sigma-Aldrich). Unless otherwise stated, chemicals for the respiration media were acquired from Scharlau Chemicals, Barcelona, Spain. After removing the papillae, most of the cortices and outer medullae was dissected from the kidneys and placed in a falcon tube on ice containing respiration media. The obtained tissue was weighed before it was minced through the metallic mesh of a tea strainer and incubated in 10 mL respiration media containing 0.75 mg/mL collagenase (Sigma-Aldrich) at 37°C for 55 min. The solution was equilibrated with a gas mixture of 95% O2/5% CO2 and manually agitated every 3–5 min throughout the incubation period. Following incubation, the solution was cooled on ice for 10 min and then filtered through a series of cell strainers of decreasing pore sizes (100, 70, and 40 μm). The filtrate was centrifuged at 100 × g for 4 min and resuspended in 5 mL respiration medium at least three times. Following the last centrifugation, the cells were resuspended in a suitable volume and quantified using a hemocytometer. Cell number and viability was determined using a trypan blue exclusion test. Hyperpolarization Hyperpolarization was performed with a SPINLAB polarizer (GE healthcare, USA). A volume of 100 μL 14.11 M [1-13C]pyruvic acid (Cambridge Isotope Laboratories, Andover, MA) containing 15 mM of the trityl radical (AH111501) (GE Healthcare, Brøndby, DK) was placed in the polarizer. The solution was polarized for three time constants (2 h) at 193.93 GHz. The hyperpolarized sample was dissolved in 17.5 mL dissolution buffer (100 mg/L EDTA) and neutralized in 1.46 mL neutralization buffer (0.4 M TRIS, 100 mg/L EDTA, 0.72 M NaOH) yielding 61.4–68.4 mM (variability due to loss in tubes) isotonic [1-13C]pyruvate at physiological pH. The sample temperature after dissolution was 30–35°C. A volume of 0.5 mL was diluted in 9.5 mL respiration media to a final concentration of approximately 10 mM [1-13C]pyruvate. Nuclear MR Spectroscopy The cell suspension was centrifuged at 100 × g for 4 min, resuspended in a volume of 200 μL, and transferred to a shortened 5 mm NMR DURAN® tube (Hounisen Laboratorieudstyr, Stilling, DK) which was placed in a custom-made coil inside an Agilent 9.4 T MR-scanner (Agilent, Santa Clara, CA). The temperature inside the scanner was kept at 37°C. A droplet of [1-13C]pyruvate with an approximate volume of 30 μL in respiration media was mixed with the cell suspension via injection through a tube. A single 4° hard pulse, 20 kHz spectral width and 4096 points NMR experiment with 180 repetitions and a repetition time of 1 s was initiated just before injection. Tubular cells (n = 3) without intervention as well as cells treated with either ouabain (n = 2; 1 mM for 15 min) to block the active electrolyte transport by means of the Na/K-ATPase or antimycin A (n = 2; 2.5 µM for 10 min.) to block the mitochondrial electron transport chain were analyzed. Data Analysis Free induction decays obtained from the MRS scans were analyzed with the software iNMR (Nucleomatica, Molfetta, Italy). The free induction decays were Fourier transformed using 8192 points and 10 Hz exponential broadening, yielding the frequency spectra. A ratiometric analysis was performed on the sum of all 180 acquisitions. This ratio has previously been shown to correlate with the apparent rate constants, with kinetic models of the rate of enzymatic conversion 19. The infusion precision of pyruvate has a significant effect on the evaluated ratios and thus the more appropriate sum of derivatives (lactate, alanine and bicarbonate) was used. The kinetic curves were visually examined in the 2D representation (for representation purposes a kinetic fit is shown) as seen on the left hand side of the 2D representation, while the sum of all spectra is depicted on the top (Fig. 2). RESULTS Cell Isolation Results from control cell isolations from 1.8 ± 0.1 g of renal tissue are depicted in Table 1. The average amount of isolated live renal tubular cells was 13.1 million with a viability of 67% (Fig. 1). Figure 1Open in figure viewerPowerPoint Trypan blue staining. Photograph taken with a microscope camera of cells from an isolation stained with trypan blue in the hemocytometer, with arrows indicating different elements. The gridlines of the hemocytometer are out of focus, as they are on another focal plane than the cells. The blue hue of the image is starkly more pronounced compared with the view through the microscope. Arrows: Examples of live RTCs (black arrows), dead RTCs (red arrows), and RBCs (white arrows). Location of the gridlines (green arrows). Table 1. Data from Four Isolation Procedures of Renal Tubular Cells for Control Experimentsa Isolation # 1 2 3 4 Mean Live renal tubular cells (x106) 11.4 15.4 16.9 8.6 13.1 ± 3.3 Viability (%) 63 65 66 75 68 ± 5 Red blood cells (x106) 6.7 1.5 3.6 1.0 3.2 ± 2.2 Red blood cell-renal tubular cell ratio 0.59 0.10 0.21 0.12 0.25 ± 0.20 a Data presented as mean ± SD. A set of control experiments was performed with pre and post viability test after 17 and 30 min showing a reduction of 4% and 21%, respectively, under similar conditions as in the hyperpolarized experiments. The amount of contaminating red blood cells was ∼5–10 times lower than that of renal tubular cells with the exception of the first isolation, which contained a relatively high amount of red blood cells (∼1.7 times lower than that of tubular cells). The lower cell yield of the fourth isolation was a result of additional rounds of centrifugation and resuspension to remove the red blood cells. Results from cell isolations for intervention experiments from 1.7 ± 0.3 g of renal tissue are depicted in Table 2. For ouabain intervention, the average amount of isolated live renal tubular cells was 15.6 million with a viability of 74%. The amount of contaminating red blood cells was ∼2.5–3 times lower than that of renal tubular cells. For antimycin A intervention, the average amount of isolated live renal tubular cells was 15.4 million with a viability of 76%. The amount of contaminating red blood cells was ∼3 times lower than that of renal tubular cells. Table 2. Data from Four Isolation Procedures of Renal Tubular Cells for Intervention Experimentsa Isolation # Ouabain Antimycin A 1 2 Mean 1 2 Mean Live renal tubular cells (x106) 15.1 16.0 15.6 16.4 14.4 15.4 Viability (%) 71 76 74 79 73 76 Red blood cells (x106) 5.0 7.0 6.0 4.8 4.0 4.4 Red blood cell-renal tubular cell ratio 0.33 0.44 0.39 0.29 0.28 0.29 a Data presented as mean. MRS A typical hyperpolarized 13C-spectrum from MRS of the isolated cell suspensions (Fig. 2), demonstrating the signal from pyruvate (173 ppm), pyruvate hydrate (181 ppm), lactate (185 ppm), bicarbonate (163 ppm) and alanine (178 ppm). The transient signal intensity shows the pyruvate and pyruvate hydrate signals occurring before the conversion to lactate, alanine and CO2/bicarbonate. The metabolic turnover was significantly altered with the treatment of ouabain or antimycin A showing increased lactate production (anaerobic) and reduced alanine (amino acid) and bicarbonate production (aerobic) (Fig. 3). The control condition experiment 2 was excluded due to an erroneous hyperpolarized experiment. Figure 2Open in figure viewerPowerPoint Hyperpolarized 13C-spectra. MR spectroscopy was performed on a cell suspension of isolated renal tubular cells administered hyperpolarized [1-13C]pyruvate, acquiring 180 13C-spectra over the course of three min. Left: The sum of all spectra acquired chronologically over 180 s. Right: Kinetic curves showing the appearance of pyruvate/20 (blue line), lactate (red line). Figure 3Open in figure viewerPowerPoint Conversion of 1-13C pyruvate to lactate, alanine and bicarbonate during control conditions and after inhibition of active tubular electrolyte transport (1 mM ouabain) or inhibition of the electron transport chain (2.5 µM antimycin A). DISCUSSION In this study, we investigated whether hyperpolarized MRS can detect metabolic markers in cell suspensions ex vivo, and our results reveal signal from lactate, bicarbonate and alanine—markers of LDH, PDH and ALT activity, respectively. The alanine and bicarbonate signals were inherently low, and the kinetic information is, therefore, likely to be prone to errors. Thus, the summed time series has to be considered the most reliable quantification measure in this study. The first cell isolation yielded a considerable amount of contaminating red blood cells compared with the rest, which may influence the results, as red blood cells are metabolically active and capable to produce both lactate and alanine from pyruvate 7, 20. However, similar signal distribution was found with low red blood cells content making it unlikely that the signal is attributed to red blood cell metabolism alone, although they may contribute. Red blood cells lack mitochondria and are not able to perform oxidative phosphorylation 7; hence, red blood cell metabolism cannot contribute to the detected bicarbonate signal. The interventional differences observed between aerobic (absent oxidative phosphorylation as antimycin A effectively blocks complex III in the electron transport chain) and reduced overall energy demand (oubain inhibits electrolyte transport by means of the Na+/K+-ATPase which is the major energy consuming process in renal tubular cells) highlights the potential for investigating the metabolic alterations in primary cells isolated from renal biopsies. This current experimental setup is limited by being an acute experiment, with no perfusion system providing new nutrients and gas supply to the cells, and secondly the cell viability with trypan blue was only investigated before the NMR experiment. The experimental design partly overcomes this limitation by imposing similar time frames for both control and metabolic modulated cells. The current setup is, however, believed to be either under hypoxic or even anoxic conditions before injection of the hyperpolarized tracer due to the high oxygen consumption in the proximal tubule cells 21. Future development of this method would benefit greatly from a perfusion system and viability measurements such as 31P NMR 11, 22. Experimental NMR limitations include high relaxation effects due to the high magnetic field (9.4T), B0 and B1 inhogeneity. The relaxation rate is faster at higher field and thus impose a direct constrain on the metabolic fate that can be interrogated 23. The combination of a horizontal MRI magnet (limiting B0) and a 5-mm solenoid coil (limiting B1) is less optimized for NMR experiments 24, and thus limits the sensitivity of experiments compared with conventional NMR systems 11. These limitations can largely be overcome by reducing the field strength and setting up the method in a dedicated NMR system 11, 12, 18, 22. Having a technique to investigate metabolism in cells ex vivo presents some advantages over cultured cells in vitro. As it operates on a timescale of hours, this approach is much faster compared with the days it takes to culture cells in vitro. Furthermore, the isolated cells are more likely to retain an in vivo-like phenotype compared with cultured cells which are generally considered to gradually undergo a phenotypical alteration 25, 26. This may alter the character and metabolism of the cultured cells and, therefore, poorly reflect the in vivo situation. This could, however, also be the case for cells ex vivo because these too are removed from their in vivo environment, but the shorter timeframe may limit the alterations and still provide a diagnostically relevant representation of the in vivo state. The present methodological study highlights the potential for metabolic phenotyping of renal tubular cells even from cells obtained by routine kidney biopsies. 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